Periaqueductal efferents to dopamine and GABA neurons of the VTA
January
Periaqueductal efferents to dopamine and GABA neurons of the VTA
Niels R. Ntamati 0 2 3
Meaghan Creed 0 2 3
Ridouane Achargui 0 2 3
Christian LuÈ scher 0 1 2 3
0 a Current address: Department of Physiology, University of Bern , Bern , Switzerland. ¤ b Current address: Department of Pharmacology, University of Maryland School of Medicine , Baltimore, MD , United States of America
1 Department of Clinical Neurosciences, Geneva University Hospital , Geneva , Switzerland
2 Department of Basic Neurosciences, Faculty of Medicine, University of Geneva , Geneva , Switzerland
3 Editor: Allan Siegel, University of Medicine & Dentistry of NJ - New Jersey Medical School , UNITED STATES
Neurons in the periaqueductal gray (PAG) modulate threat responses and nociception. Activity in the ventral tegmental area (VTA) on the other hand can cause reinforcement and aversion. While in many situations these behaviors are related, the anatomical substrate of a crosstalk between the PAG and VTA remains poorly understood. Here we describe the anatomical and electrophysiological organization of the VTA-projecting PAG neurons. Using rabies-based, cell type-specific retrograde tracing, we observed that PAG to VTA projection neurons are evenly distributed along the rostro-caudal axis of the PAG, but concentrated in its posterior and ventrolateral segments. Optogenetic projection targeting demonstrated that the PAG-to-VTA pathway is predominantly excitatory and targets similar proportions of Ih-expressing VTA DA and GABA neurons. Taken together, these results set the framework for functional analysis of the interplay between PAG and VTA in the regulation of reward and aversion.
Introduction
The periaqueductal gray (PAG) is a heterogeneous midbrain structure that is critical for the
endogenous modulation of nociception and for the expression of defensive behaviors [
1
].
These functions have been shown to be mediated by neurons anatomically segregated in the
longitudinal columns corresponding to the dorsolateral (dl), lateral (l) and ventrolateral (vl)
subdivisions of the PAG [
2
]. The PAG is also the major site of action for opioid analgesia [
3,4
].
Anatomical tracing studies have described ascending and descending projections from the
PAG to a variety of brain structures [
5
]. Among these projection targets is the ventral
tegmental area (VTA), a major component of the brain reward system [
6
]. While it is primarily
known for its role in reward prediction, and positive reinforcement [
7,8
], the VTA has also
been implicated in the modulation of nociception and in the expression of fear and aversive
responses [9±12]. The engagement of VTA dopamine- (DA) and gamma-aminobutyric acid
(GABA)-releasing neurons with PAG afferents through both symmetric and asymmetric
synaptic contacts has been demonstrated with rabies-assisted retrograde tracing and
ultrastructural immunoelectron microscopic analyses [
13,14
].
Competing interests: The authors have declared
that no competing interests exist.
It remains elusive, however, whether the input neurons providing these VTA afferents
spatially segregate within specific PAG columns, potentially associating the PAG-to-VTA pathway
with specific anti-nociceptive or defensive functions. Moreover, evidence for an
electrophysiologically functional connection, and knowledge of its excitatory or inhibitory effect onto VTA
DA and GABA neurons is still lacking. To this end, the present study will describe the
rostrocaudal distribution of VTA-projecting neurons across the PAG columns, and will test whether
these neurons exhibit a preferential excitatory or inhibitory effect on DA and GABA neurons
of the VTA.
Materials and methods
Animal procedures and ethics statement
Experiments were performed on DAT-Cre [
15
] and GAD65-Cre mice [
16
] of both sexes. All
animal procedures were performed in accordance with the authors' university animal care
committee's regulations and approved by the animal welfare commission of the Canton of
Geneva (approval number GE-71-16). At the conclusion of the experiments, animals were
euthanized with an anesthetic overdose of pentobarbital (300 mg/kg i.p., Streuli Pharma) or by
decapitation under isoflurane anesthesia (2±5%, Attane).
Injection procedures
All stereotaxic intracranial injections were performed under isoflurane anesthesia (2±5%)
using glass capillary pipettes connected to a microinjection pump (Narishige) at a rate of ~100
nl min-1. The coordinates used were (from bregma, in mm): AP -3.4, ML ±0.5, DV -4.3 for
VTA injections; AP -4.0, ML ±0.3, DV -2.6 for PAG injections. For retrograde tracing
experiments, 300 nl of a 1:1 mixture of AAV8-CAG-DIO-RG (2.5 × 1012 IU/ml) and
AAV5-EF1aDIO-TVA-mCherry (4.9 × 1012 IU/ml) was injected unilaterally in the VTA, followed 2 weeks
later by the injection of 800 nl of RVΔG-EnvA-EGFP (107 IU/ml) at the same coordinates. For
patch clamp experiments, animals were bilaterally injected with AAV5-EF1a-D (...truncated)